Expression Of Pulmonary Surfactant-associated Protein A In Rats After Freshwater Drowning

ObjectiveDrowning accidents are not uncommon. Drowning is the second leading cause of unintentional injury deaths around world. So to study the mechanism of drowning is important for early treatment.To establish an animal model of ALI and observe the changes of respiratory rate(RR),blood-gas,pulmonary tissue pathology after freshwater drowning. The expression of SP-A was detected after drowning for 2, 4, 6 hours respectively. To investigate the mechanism of ALI after freshwater drowning and provide new thinking for clinical treatment.Material and methods1. Study groupsTwenty-four Sprague-Dawley rats(weight 205±10g) were from Fourth Military Medical University. Animal were randomly divided into two groups: control group(n=6) and drowning group(n=18). The drowning group were furthly divdied into subgroups: 2h group,4h group,6h group.2. Establish the model of lung injury after freshwater drowning Rats were anaesthetized with 25 % ethylcarbamate(5 ml/Kg) by peritoneal injection. Rats were maintained in the supine position during experiments. A heparin-filled catheter was inserted into the right carotid artery for withdrawal of references blood flow samples. Tracheostomy was performed, and a catheter was advanced to a position approximately 1 cm above the carina. Following a 20 min stable baseline period, then using 10 ml syringe, 8 ml/Kg body weight of feshwater was instilled with 1 min into both lungs.3. MethodRR were recorded and the blood of carotid arterial was taken to test blood gas. RR and Arterial blood gas analyses were performed 0 min before and 5 min after freshwater instillation and then every two hours during the 6 h study period. Rats were sacrificed 2, 4, 6h after drowning respectively. For light microscopic exmination, the left lower lung was fixed in 10% formalin, and then embedded in paraffin wax, sliced, and stained with hematoxylin-eosin. Meanwhile the expression of SP-A was detected by immunohistochemistry. The right lower lung tissue was snap-frozen in liquid nitrogen and stored at -70℃. Reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate relative amount of SP-AmRNA expression.Results1. General state and RRCompared with the control group, the respiratory rates accelerated significantly, then slowed gradually but always faster than normal range in the drowning group. After instillation, part of rats showed unregular RR and had tic of limbs, and foam liquid exuded from trachea.2. Arterial blood gas analysisPaO₂ decreased significantly 5 min after instillation then elevated but remained at a low level. PaCO2 increased significantly 5 min after instillation then decreased and remained below normal level.3. Histopathological findingsFresh water drowning caused direct and indirect lung injury. In the drowning group, alveolar wall collapse, congestion and thickening of the alveolar-capillary membrane, alveolar and interstitial edema and focal atelectasis were observed. PMN infiltration was significantly more severe in drowning rats compared with control groups. PMN infiltration progressed with the time in the drowning group.4. Immunostaining of pulmonary SP-A and the mRNA level of SP-AIn the control group, SP-A was distributed in a consecutive linear fashion on the intra-alveolar interior surface, and part of the alveolar cells were also observed SP-A granular staining. In drowning group, SP-A was demonstrated on the alveolar interior surface and the interface of the intra-alveolar effusion in a granular deposit fashion with the quantity decreased gradually compared to the control group. The SP-A mRNA level of lung in drowning group at every time points was significantly lower than that of in the control group and decreased obviously 2 h after drowning , then showed a rising tendency, but maintained a lower level.ConclusionFreshwater drowning might result in lung injure and abnormalities in the quality and quantity of SP-A.The level of SP-A in lung decreased ,which maybe due to the injury of alveolar epithelial cell in early stage, then maybe related to the increase of pulmonary capiIlary permeability.